Human theta subunit of the GABAa receptor

ABSTRACT

This invention concerns the cloning of a novel cDNA sequence encoding a particular subunit of the human GABA A  receptor. In addition, the invention relates to a stable cell line capable of expressing said cDNA and to the use of the cell line in a screening technique for the design and development of subtype-specific medicaments.

This specification claims the benefit of PCT/GB/01206, filed Apr. 24,1998, and GB Application 9708479.2, filed Apr. 25, 1997.

This invention concerns the cloning of a novel cDNA sequence encoding aparticular subunit of the human GABA_(A) receptor. In addition, theinvention relates to a stable cell line capable of expressing said cDNAand to the use of the cell line in a screening technique for the designand development of subtype-specific medicaments.

Gamma-amino butyric acid (GABA) is a major inhibitory neurotransmitterin the central nervous system. It mediates fast synaptic inhibition byopening the chloride channel intrinsic to the GABA_(A) receptor. Thisreceptor comprises a multimeric protein of molecular size 230-270 kDawith specific binding sites for a variety of drugs includingbenzodiazepines, barbiturates and β-carbolines, in addition to sites forthe agonist ligand GABA (for reviews see MacDonald and Olsen, Ann. Rev.Neurosci., 1994, 17, 569; and Whiting et al, Int. Rev. Neurobiol., 1995,38, 95).

Molecular biological studies demonstrate that the receptor is composedof several distinct types of subunit, which are divided into fourclasses (α, β, γ and δ) based on their sequence similarities. To date,in mammals, six types of α (Schofield et al., Nature (London), 1987,328, 221; Levitan et al., Nature (London), 1988, 335, 76; Ymer et al.,EMBO J., 1989, 8, 1665; Pritchett & Seeberg, J. Neurochem., 1990, 54,802; Luddens et al., Nature (London), 1990, 346, 648; and Khrestchatiskyet al., Neuron, 1989, 3, 745), three types of β (Ymer et al., EMBO J.,1989, 8, 1665), three types of γ (Ymer et al., EMBO J., 1990, 9, 3261;Shivers et al., Neuron, 1989, 3, 327: and Knoflach et al, FEBS Lett.,1991, 293, 191) and one δ subunit (Shivers et al., Neuron, 1989, 3, 327)have been identified. More recently, a further member of the GABAreceptor gene family, ε, has been identified (Davies et al, Nature,1997, 385, 820). The polypeptide is 506 amino acids in length andexhibits greatest amino acid sequence identity with the GABA_(A)receptor γ₃ subunit (47%), although this degree of homology is notsufficient for it to be classified as a fourth γ subunit.

The differential distribution of many of the subunits has beencharacterised by in situ hybridisation (Shivers et al., Neuron, 1989, 3,327; Wisden et al, J. Neurosci., 1992, 12, 1040; and Laurie et al, J.Neurosci, 1992, 12, 1063) and this has permitted it to be speculatedwhich subunits, by their co-localisation, could theoretically exist inthe same receptor complex.

Various combinations of subunits have been co-transfected into cells toidentify synthetic combinations of subunits whose pharmacology parallelsthat of bona fide GABA_(A) receptors in vivo (Pritchett et al., Science,1989, 245, 1389; Pritchett and Seeberg, J. Neurochem., 1990, 54, 1802;Luddens et al., Nature (London), 1990, 346, 648; Hadingham et al, Mol.Pharmacol., 1993, 43, 970; and Hadingham et al., Mol. Pharmacol., 1993,44, 1211). This approach has revealed that, in addition to an α and βsubunit, either γ₁ or γ₂ (Pritchett et al., Nature (London), 1989, 338,582; Ymer et al., EMBO J., 1990, 9, 3261; and Wafford et al., Mol.Pharmacol., 1993, 44, 437) or γ₃ (Herb et al., Proc. Natl. Acad. Sci.USA, 1992, 89, 1433; Knoflach et al., FEBS Lett., 1991, 293, 191; andWilson-Shaw et al., FEBS Lett., 1991, 284, 211) is also generallyrequired to confer benzodiazepine sensitivity, and that thebenzodiazepine pharmacology of the expressed receptor is largelydependent on the identity of the α and γ subunits present. Receptorscontaining a δ subunit (i.e. αγδ) do not appear to bind benzodiazepines(Shivers et al., Neuron, 1989, 3, 327; and Quirk et al., J. Biol. Chem.,1994, 269, 16020). Combinations of subunits have been identified whichexhibit the pharmacological profile of a BZ₁ type receptor (α₁β₁γ₂) anda BZ₂ type receptor (α₂β₁γ₂ or α₃β₁γ₂, Pritchett et al., Nature(London), 1989, 338, 582), as well as GABA_(A) receptors with a novelpharmacology, α₅β₂γ₂ (Pritchett and Seeberg, J. Neurochem., 1990, 54,1802), α₄β₂γ₂ (Wisden et al, FEBS Lett., 1991, 289, 227) and α₆β₂γ₂(Luddens et al., Nature (London), 1990, 346, 648). The pharmacology ofthese expressed receptors appears similar to that of those identified inbrain tissue by radioligand binding, and biochemical expperiments havebegun to determine the subunit composition of native GABA receptors(McKernan & Whiting, Tr. Neurosci., 1996, 19, 139). The exact structureof receptors in vivo has yet to be definitively elucidated.

The present invention relates to a new class of GABA receptor subunit,hereinafter referred to as the theta subunit (θ subunit).

The nucleotide sequence for the theta subunit, together with its deducedamino acid sequence corresponding thereto, is depicted in FIG. 1 of theaccompanying drawings.

The present invention accordingly provides, in a first aspect, a DNAmolecule encoding the theta subunit of the human GABA receptorcomprising all or a portion of the sequence depicted in FIG. 1, or amodified human sequence.

In an alternative aspect, the present invention provides a DNA moleculeencoding the theta subunit of the human GABA receptor comprising all ora portion of the sequence depicted in FIG. 2, or a modified humansequence.

The term “modified human sequence” as used herein referes to a variantof the DNA sequences depicted in FIG. 1 and FIG. 2. Such variants may benaturally occuring allelic variants or non-naturally occuring or“engineered” variants. Allelic variation is well known in the art inwhich the nucleotide sequence may have a substitution, deletion oraddition of one or more nucleotides without substantial alteration ofthe function of the encoded polypeptide. Particularly preferred allelicvariants arise from nucleotide substitution based on the degeneracy ofthe genetic code.

The sequencing of the novel cDNA molecules in accordance with theinvention can conveniently be carried out by the standard proceduredescribed in accompanying Example 1; or may be accomplished byalternative molecular cloning techniques which are well known in theart, such as those described by Maniatis et al. in Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, New York, 2nd edition,1989.

In a further aspect, the present invention also relates topolynucleotides (for example, cDNA, genomic DNA or synthetic DNA) whichhybridize under stringent conditions to the DNA molecules depicted inFIG. 1 and FIG. 2. As used herein, the term “stringent conditions” willbe understood to require at least 95% and preferably at least 97%identity between the hybridized sequences. Polynucleotides whichhybridize under stringent conditions to the DNA molecules depicted inFIG. 1 and FIG. 2 preferably encode polypeptides which exhibitsubstantially the same biological activity or function as thepolypeptides depicted in FIG. 1 and FIG. 2, respectively.

The present invention further relates to a GABA theta subunitpolypeptide which has the deduced amino acid sequence of FIG. 1 or FIG.2, as well as fragments, analogs and derivatives thereof.

The terms “fragment”, “derivative” and “analog” when referring to thepolypeptide of FIG. 1 or FIG. 2, means a polypeptide which retainsessentially the same biological activity or function as the polypeptidedepicted in FIG. 1 or FIG. 2. Thus, an analog may be, for example, aproprotein which can be activated by cleavage of the proprotein portionto produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 or FIG.2 may be one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residues may or may not be one encoded by the genetic code; or onein which one or more of the amino acid residues includes a substituentgroup; or one in which the mature polypeptide is fused with anothercompound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol); or one in which theadditional amino acids are fused to the mature polypeptide, such as aleader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the technicalcapabilities of those skilled in the art.

The polypeptides and DNA molecules of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring DNA molecule orpolypeptide present in a living animal is not isolated, but the same DNAmolecule or polypeptide, separated from some or all of the coexistingmaterials in the natural system, is isolated. Such DNA molecules couldbe part of a vector and/or such DNA molecules or polypeptides could bepart of a composition, and still be isolated in that such vector orcomposition is not part of its natural environment.

In another aspect, the invention provides a recombinant expressionvector comprising the nucleotide sequence of the human GABA receptortheta subunit together with additional sequences capable of directingthe synthesis of the said human GABA receptor theta subunit in culturesof stably co-transfected eukaryotic cells.

The term “expression vectors” as used herein refers to DNA sequencesthat are required for the transcription of cloned copies of recombinantDNA sequences or genes and the translation of their mRNAs in anappropriate host. Such vectors can be used to express eukaryotic genesin a variety of hosts such as bacteria, blue-green algae, yeast cells,insect cells, plant cells and animal cells. Specifically designedvectors allow the shuttling of DNA between bacteria-yeast,bacteria-plant or bacteria-animal cells. An appropriately constructedexpression vector should contain: an origin of replication forautonomous replication in host cells, selective markers, a limitednumber of useful restriction enzyme sites, a high copy number, andstrong promoters. A promoter is defined as a DNA sequence that directsRNA polymerase to bind to DNA and to initiate RNA synthesis. A strongpromoter is one which causes mRNAs to be initiated at high frequency.Expression vectors may include, but are not limited to, cloning vectors,modified cloning vectors, specifically designed plasmids or viruses.

The term “cloning vector” as used herein refers to a DNA molecule,usually a small plasmid or bacteriophage DNA capable of self-replicationin a host organism, and used to introduce a fragment of foreign DNA intoa host cell. The foreign DNA combined with the vector DNA constitutes arecombinant DNA molecule which is derived from recombinant technology.Cloning vectors may include plasmids, bacteriophages, viruses andcosmids.

The recombinant expression vector in accordance with the invention maybe prepared by inserting the nucleotide sequence of the GABA thetasubunit into a suitable precursor expression vector (hereinafterreferred to as the “precursor vector”) using conventional recombinantDNA methodology known from the art. The precursor vector may be obtainedcommercially, or constructed by standard techniques from knownexpression vectors. The precursor vector suitably contains a selectionmarker, typically an antibiotic resistance gene, such as the neomycin orampicillin resistance gene. The precursor vector preferably contains aneomycin resistance gene, adjacent the SV40 early splicing andpolyadenylation region; an ampicillin resistance gene; and an origin ofreplication, e.g. pBR322 ori. The vector also preferably contains aninducible promoter, such as MMTV-LTR (inducible with dexamethasone) ormetallothionin (inducible with zinc), so that transcription can becontrolled in the cell line of this invention. This reduces or avoidsany problem of toxicity in the cells because of the chloride channelintrinsic to the GABA_(A) receptor.

One suitable precursor vector is pMAMneo, available from ClontechLaboratories Inc. (Lee et al., Nature, 1981, 294, 228; and Sardet etal., Cell, 1989, 56, 271). Alternatively the precursor vector pMSGneocan be constructed from the vectors pMSG and pSV2neo.

The recombinant expression vector of the present invention is thenproduced by cloning the GABA receptor theta subunit cDNA into the aboveprecursor vector. The receptor subunit cDNA is subcloned from the vectorin which it is harboured, and ligated into a restriction enzyme site,e.g. the Hind III site, in the polylinker of the precursor vector, forexample pMAMneo or pMSGneo, by standard cloning methodology known fromthe art, and in particular by techniques analogous to those describedherein. Before this subdoning, it is often advantageous, in order toimprove expression, to modify the end of the theta subunit cDNA withadditional 5′ untranslated sequences, for example by modifying the 5′end of the theta subunit DNA by addition of 5′ untranslated regionsequences from the α₁ subunit DNA. Alternatively, expression of thetheta subunit cDNA may be modified by the insertion of an epitope tagsequence such as c-myc.

According to a further aspect of the present invention, there isprovided a stably co-transfected eukaryotic cell line capable ofexpressing a GABA receptor, which receptor comprises the theta receptorsubunit, at least one alpha receptor subunit and optionally one or morebeta, gamma, delta, or epsilon receptor subunit.

This is achieved by co-transfecting cells with multiple expressionvectors, each harbouring cDNAs encoding for an α, θ, and optionally oneor more β, γ, δ , or GABA receptor subunits. In a further aspect,therefore, the present invention provides a process for the preparationof a eukaryotic cell line capable of expressing a GABA receptor, whichcomprises stably co-transfecting a eukaryotic host cell with at leasttwo expression vectors, one such vector harbouring the cDNA sequenceencoding the theta GABA receptor subunit, and another such vectorharbouring the cDNA sequence encoding an alpha GABA receptor subunit.The stable cell-line which is established expresses an αθ GABA receptor.

Each receptor thereby expressed, comprising a unique combination of α, θand optionally one or more subunits selected from β, γ, δ or δ subunits,will be referred to hereinafter as a GABA receptor “subunitcombination”.

Expression of the GABA receptor may be accomplished by a variety ofdifferent promoter-expression systems in a variety of different hostcells. The eukaryotic host cells suitably include yeast, insect andmammalian cells. Preferably the eukaryotic cells which can provide thehost for the expression of the receptor are mammalian cells. Suitablehost cells include rodent fibroblast lines, for example mouse Ltk⁻,Chinese hamster ovary (CHO) and baby hamster kidney (BHK); HeLa; andHEK293 cells. It is necessary to incorporate at least one a subunit, theθ subunit, and optionally one or more subunits selected from β, γδ or δinto the cell line in order to produce the required receptor. Withinthis limitation, the choice of receptor subunit combination is madeaccording to the type of activity or selectivity which is being screenedfor.

In order to employ this invention most effectively for screeningpurposes, it is preferable to build up a library of cell lines, eachwith a different combination of subunits. Typically a library of 5 or 6cell line types is convenient for this purpose. Preferred subunitcombinations include: αθβ, αθγ, αθδ, and αθε, and most especially α₁θγ₂.Further preferred subunit combinations include αβθγ and αβθε, and mostespecially α₂β₁θγ₁ and α₂β₃θγ₂.

Cells are then co-transfected with the desired combination of theexpression vectors. There are several commonly used techniques fortransfection of eukaryotic cells in vitro. Calcium phosphateprecipitation of DNA is most commonly used (Bachetti et al., Proc. Natl.Acad. Sci. USA, 1977, 74, 1590-1594; Maitland et al., Cell, 1977, 14,133-141), and represents a favoured technique in the context of thepresent invention.

A small percentage of the host cells takes up the recombinant DNA. In asmall percentage of those, the DNA will integrate into the host cellchromosome. Because an antibitotic resistance marker gene, such as theneomycin or zeocin resistance gene, will have been incorporated intothese host cells, they can be selected by isolating the individualclones which will grow in the presence of the chosen antibiotic, e.g.neomycin or zeocin. Each such clone may then tested to identify thosewhich will produce the receptor. This may be achieved by inducing theproduction, for example with dexamethasone, and then detecting thepresence of receptor by means of radioligand binding.

Alternatively, expression of the GABA receptor may be effected inXenopus oocytes (see, for instance, Hadingham et al. Mol. Pharmacol.,1993, 44, 1211-1218). Briefly, isolated oocyte nuclei are injecteddirectly with injection buffer or sterile water containing at least onealpha subunit, the theta subunit, and optionally one or more beta,gamma, delta or epsilon receptor subunits, engineered into a suitableexpression vector. The oocytes are then incubated.

The expression of subunit combinations in the transfected oocytes may bedemonstrated using conventional patch clamp assay. This assay measuresthe charge flow into and out of an electrode sealed on the surface ofthe cell. The flow of chloride ions entering the cell via the GABA gatedion channel is measured as a function of the current that leaves thecell to maintain electrical equilibrium within the cell as the gateopens.

In a further aspect, the present invention provides protein preparationsof GABA receptor subunit combinations, especially human GABA receptorsubunit combinations, derived from cultures of stably transfectedeukaryotic cells.

The protein preparations of GABA receptor subunit combinations can berecovered and purified from recombinant cell cultures by methodsincluding ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

The GABA theta subunit polypeptide of the present invention is alsouseful for identifying other subunits of the GABA receptor. An exampleof a procedure for identifying these subunits comprises raising hightitre polyclonal antisera against unique, bacterially expressed GABAtheta polypeptides. These polyclonal antisera are then used toimmunoprecipitate detergent-solubilized GABA receptors from a mammalianbrain, for example, a rat brain.

The invention also provides preparations of membranes containing subunitcombinations of the GABA receptor, especially human GABA receptorsubunit combinations, derived from cultures of stably transfectedeukaryotic cells.

The cell line, and the membrane preparations therefrom, according to thepresent invention have utility in screening and design of drugs whichact upon the GABA receptor, for example benzodiazepines, barbiturates,β-carbolines and neurosteroids.

Receptor localisation studies using in situ hybridization in monkeybrains shows that the θ subunit has a restricted localisation; residingmainly in components of the limbic system (involved in emotions such asrage, fear, motivation sexual behaviours and feeding); medial septum,cingulate cortex, the amygdala and hippocampal fields, in varioushypothalamic nuclei, and in regions that have been associated with painperception; the cingulate cortex, insular cortex, and in mid brain andpons structures.

The present invention accordingly provides the use of stablycotransfected cell lines described above, and membrane preparationsderived therefrom, in screening for and designing medicaments which actupon GABA receptors comprising the θ subunit. Of particular interest inthis context are molecules capable of interacting selectively with GABAreceptors made up of varying subunit combinations. As will be readilyapparent, the cell line in accordance with the present invention, andthe membrane preparations derived therefrom, provide ideal systems forthe study of structure, pharmacology and function of the various GABAreceptor subtypes. In particular, preferred screens are functionalassays utilizing the pharmacological properties of the GABA receptorsubunit combinations of the present invention.

Thus, according to a further aspect of the present invention, there isprovided a method for determining whether a ligand, not known to becapable of binding to a human GABA_(A) receptor comprising the thetasubunit, can bind to a human GABA_(A) receptor comprising the thetasubunit, which comprises contacting a mammalian cell comprising DNAmolecules encoding at least one alpha receptor subunit, the thetareceptor subunit, and optionally one or more beta, gamma, delta orepsilon receptor subunits with the ligand under conditions permittingbinding of ligands known to bind to the GABA_(A) receptor, detecting thepresence of any of the ligand bound to the GABA_(A) receptor comprisingthe theta subunit, and thereby determining whether the ligand binds tothe GABA_(A) receptor comprising the theta subunit. The thetasubunit-encoding DNA in the cell may have a coding sequencesubstantially the same as the coding sequence shown in FIG. 1 or FIG. 2.Preferably, the mammalian cell is non-neuronal in origin. An example ofa non-neuronal mammalian cell is a fibroblast cell such as an Ltk⁻cell.The preferred method for determining whether a ligand is capable ofbinding to a human GABA_(A) receptor comprising the theta subunitcomprises contacting a transfected non-neuronal mammalian cell (i.e. acell that does not naturally express any type of GABA_(A) receptor, andthus will only express such a receptor if it is transfected into thecell) expressing a GABA_(A) receptor comprising the theta subunit on itssurface, or contacting a membrane preparation from such a transfectedcell, with the ligand under conditions which are known to prevail, andthus to be associated with, in vivo binding of the ligands to a GABA_(A)receptor comprising the theta subunit, detecting the presence of any ofthe ligand being tested bound to the GABA_(A) receptor comprising thetheta subunit on the surface of the cell, and thereby determiningwhether the ligand binds to a human GABA_(A) receptor comprising thetheta subunit. This response system may be based on ion flux changesmeasured, for example, by scintillation counting (where the ion isradiolabelled) or by interaction of the ion with a fluorescent marker.Particularly suitable ions are chloride ions. Such a host system isconveniently isolated from pre-existing cell lines. Such a transfectionsystem provides a complete response system for investigation or assay ofthe activity of human GABA_(A) receptors comprising the theta subunitwith ligands as described above. Transfection systems are useful asliving cell cultures for competitive binding assays between known orcandidate drugs and ligands which bind to the receptor and which arelabeled by radioactive, spectroscopic or other reagents. Membranepreparations containing the receptor isolated from transfected cells arealso useful for these competitive binding assays. A transfection systemconstitutes a “drug discovery system” useful for the identification ofnatural or synthetic compounds with potential for drug development thatcan be further modified or used directly as therapeutic compounds toactivate, inhibit or modulate the natural functions of human GABA_(A)receptors comprising the theta subunit. The transfection system is alsouseful for determining the affinity and efficacy of known drugs at humanGABA_(A) receptor sites comprising the theta subunit.

This invention also provides a method of screening drugs to identifydrugs which specifically interact with, and bind to, a human GABA_(A)receptor comprising the theta subunit on the surface of a cell whichcomprises contacting a mammalian cell comprising DNA molecules encodingat least one alpha receptor subunit, the theta receptor subunit andoptionally one or more beta, gamma, delta or epsilon receptor subunitson the surface of a cell with a plurality of drugs, determining thosedrugs which bind to the mammalian cell, and thereby identifying drugswhich specifically interact with, and bind to, human GABA_(A) receptorscomprising the theta subunit. The theta subunit-encoding DNA in the cellmay have a coding sequence substantially the same as the coding sequenceshown in FIG. 1 or FIG. 2. Preferably, the mammalian cell isnon-neuronal in origin. An example of a non-neuronal mammalian cell is afibroblast cell such as an Ltk⁻cell. Drug candidates are identified bychoosing chemical compounds which bind with high affinity to theexpressed GABA_(A) receptor protein in transfected cells, usingradioligand binding methods well known in the art. Drug candidates arealso screened for selectivity by identifying compounds which bind withhigh affinity to one particular GABA_(A) receptor combination but do notbind with high affinity to any other GABA_(A) receptor combination or toany other known receptor site. Because selective, high affinitycompounds interact primarily with the target GABA_(A) receptor siteafter administration to the patient, the chances of producing a drugwith unwanted side effects are minimized by this approach.

In the above screens, the mammalian cell may, for example, comprise DNAmolecules encoding at least one alpha receptor subunit, the thetasubunit, and optionally one or more gamma receptor subunits andoptionally one or more beta receptor subunits.

More preferably, in the above screens, the mammalian cell comprises DNAmolecules encoding at least one alpha receptor subunit, at least onegamma receptor subunit and the theta receptor subunit.

Ligands or drug candidates identified above may be agonists orantagonists at human GABA_(A) receptors comprising the theta subunit, ormay be agents which allosterically modulate a human GABA_(A) receptorcomprising the theta subunit. These ligands or drug candidatesidentified above may be employed as therapeutic agents, for example, forthe modulation of emotions such as rage and fear, of sexual and appetitebehaviours and of pain perception.

The ligands or drug candidates of the present invention thus identifiedas therapeutic agents may be employed in combination with a suitablepharmaceutical carrier. Such compositions comprise a therapeuticallyeffective amount of the agonist or antagonist, and a pharmaceuticallyacceptable carrier or excipient.

Preferably the compositions containing the ligand or drug candidateidentified according to the methods of the present invention are in unitdosage forms such as tablets, pills, capsules, wafers and the like.Additionally, the therapeutic agent may be presented as granules orpowders for extemporaneous formulation as volume defined solutions orsuspensions. Alternatively, the therapeutic agent may be presented inready-prepared volume defined solutions or suspensions. Preferred formsare tablets and capsules.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother pharmaceutical diluents, e.g. water, to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer which serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally include aqueoussolutions, suitably flavoured syrups, aqueous or oil suspensions, andflavoured emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil, peanut oil or soybean oil, as well as elixirs and similarpharmaceutical vehicles. Suitable dispersing or suspending agents foraqueous suspensions include synthetic and natural gums such astragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose,methylcellulose, polyvinyl-pyrrolidone or gelatin.

Compositions of the present invention may also be administered via thebuccal cavity using conventional technology, for example, absorptionwafers.

Compositions in the form of tablets, pills, capsules or wafers for oraladministration are particularly preferred.

A minimum dosage level for the ligand or drug candidate identifiedaccording to the methods of the present invention is about 0.05 mg perday, preferably about 0.5 mg per day and especially about 2.5 mg perday. A maximum dosage level for the ligand or drug candidate is about3000 mg per day, preferably about 1500 mg per day and especially about500 mg per day. The compounds are administered on a regimen of 1 to 4times daily, preferably once or twice daily, and especially once a day.

It will be appreciated that the amount of the therapeutic agent requiredfor use therapy will vary not only with the particular compounds orcompositions selected but also with the route of administration, thenature of the condition being treated, and the age and condition of thepatient, and will ultimately be at the discretion of the patient'sphysician or pharmacist.

DESCRIPTION OF FIGURES

FIG. 1: Nucleotide sequence for the theta subunit, together with itsdeduced amino acid sequence corresponding thereto (SEQ.ID.NO.1 andSEQ.ID.NO.2, respectively)

FIG. 2: Preferred nucleotide sequence for the theta subunit, togetherwith its deduced amino acid sequence corresponding thereto (SEQ.ID.NO.3and SEQ.ID.NO.4, respectively).

FIG. 3: GABA dose-response curves on HEK cells transiently transfectedwith and without θ subunit-containing GABA-A receptors (β₂β₁θγ₁ andα₂β₁γ₁).

The following non-limiting Examples illustrate the present invention.

EXAMPLE 1 ISOLATION AND SEQUENCING OF A cDNA ENCODING THE HUMAN GABA_(A)RECEPTOR θ SUBUNIT

The Genbank database was searched with GABA_(A) receptor polypeptideamino acid sequences using the BLAST searching algorithm, and a numberof homologous sequences identified. One of these U47334 was investigatedin more detail. U47334 contained sequences homologous to part of theamino-terminal extracellular domain and the TM4 spanning domain of otherGABA_(A) receptor subunits, but did not appear to contain any sequencehomologous to the regions spanning these domains. Polymerase chainreaction (PCR) was performed to determine if the size if the U47334sequence was correct, or was for example, the result of an incorrectsplicing event. For PCR, a sense (5′ gcaaatgaagctgtggttc 3′) (SEQ.ID.NO.5) and antisense (5′ caaatgttgaacaacccaaag 3′) (SEQ.ID.NO. 6) primerwere generated from the U47334 sequence, and PCR performed usingstandard conditions (Whiting et al, PNAS) using human whole brain cDNA(Clontech) as a template. A second PCR reation was then performed usingnested sense (5′ gcctgagaccgaattttgg 3′) (SEQ.ID.NO. 7) and antisense(5′ ggaaccgggaccacttgtc 3′) (SEQ.ID.NO. 8) primers generated from theU47334 sequence, and using the products from the first PCR as atemplate. A single PCR product of approximately 1600 bp was obtainedsuggesting that the U47334 sequence represents an incorrectly processedmessage. This product was sequenced directly using an Applied Biosystems373 DNA sequencer and dye terminator chemistry.

cDNA sequences 5′ and 3′ of the U47334 sequence were obtained by 5′- and3′-anchored PCR using human brain Marathon cDNA cloning kit (Clontech)according to the manufacturer's protocols. The nested antisense (5′tagtccagggtcaagttc 3′ and 5′ tagtatgctaagcgtgaatc 3′) (SEQ.ID.NOS. 9 and10) and sense (5′ gagtttgaggatagttgc 3′ and 5′ tgctccttcactgaaggg 3′)(SEQ.ID.NOS. 11 and 12) primers were derived from both the U47334sequence and the sequence from the initial PCR amplifications. The PCRproducts were sequenced directly as previously described.

A full length cDNA was generated by PCR using primers derived fromsequences downstream of the innitiating ATG (5′ccatgactcaagcttgccaccatgctgcgagccgeagtgatc 3′, incorporating a HindIIIsite) (SEQ.ID.NO. 13) and in the 3′ UT of the anchored PCR product (5′tgaaaggagcacagcacagtgctcccg 3′) (SEQ.ID.NO. 14). The PCR product (1958bp) was cloned into pMOS (Amersham), subcloned into pCDNAI Amp(Invitrogen), and sequenced completey on both strands by primer walking.Sequence analysis was performed using Inherit (Applied Biosystems),Sequencher (Genecodes), and Genetics Computer Group (Univ. Wisconsin)computer programs.

The coding region encodes 627 amino acids and has all the structuralmotifs expected of a ligand gated ion channel subunit. Comparison withother ligand gated ion channel subunits indicates that it is mostsimilar to GABA_(A) receptor subunits, the highest homology being withthe β₁ subunit (45% identity). However, this sequence identity issufficiently low as to indicate that the new subunit cannot beclassified as a fourth human β subunit, but represents a novel class ofsubunit, classified as θ, within the GABA receptor gene family.

EXAMPLE 2 LOCALISATION OF THE θ SUBUNIT IN MONKEY BRAIN BY IN SITUHYBRIDISATION

Antisense oligonucleotide probes to the human θ subunit sequence weregenerated on an Applied Biosystems Automated DNA synthesiser Probe 1 5′CTG-CTT-CTT-GCA-CAC-CCT-TCT-CGC-CAT-GGT-GAA-GCA-TGG-GCT-TCC 3′(SEQ.ID.NO. 15) Probe 25′TGT-CGC-CTA-GGC-TGG-CGC-CGA-GGT-CCT-CGA-CTG-TAG-AAA-AGA-TAG 3′(SEQ.ID.NO. 16)

Each oligonucleotide was 3′-end labelled with [³⁵S] deoxyadenosine5′-(thiotriphosphate) in a 30:1 molar ratio of³⁵S-isotope:oligonucleotide using terminal deoxynucleotidyl transferasefor 15 min at 37° C. in the reaction buffer supplied. Radiolabelledoligonucleotide was separated from unincorporated nucleotides usingSephadex G50 spin columns. The specific activities of the labelledprobes in several labelling reactions varied from 1.2-2.3×10⁹ cpm/mg.Monkey brains were removed and fresh frozen in 1 cm blocks. 12 μmsections were taken and fixed for in situ hybridisation. Hybridisationof the sections was carried out according to the method ofSirinathsingji and Dunnett (Imaging gene expression in neural graft;Molecular Imaging in Neuroscience: A Practical Approach, N. A. Sharif(ed), Oxford University Press, Oxford, pp43-70, 1993). Briefly, sectionswere removed from alcohol, air dried and 3×10⁵ cpm of each ³⁵S-labelledprobe in 100 μl of hybridisation buffer was applied to each slide.Labelled “antisense” probe was also used in the presence of an excess(100×) concentration of unlabelled antisense probe to definenon-specific hybridisation. Parafilm coverslips were placed over thesections which were incubated overnight (about 16 hr) at 37° C.Following hybridisation the sections were washed for 1 hr at 57° C. in1×SSC then rinsed briefly in 0.1×SSC, dehydrated in a series ofalcohols, air dried and exposed to Amersham Hyperfilm max X-ray film andthe relative distribution of the mRNA assessed for a variety of brainregions.

Messenger RNA for the subunit was seen in components of the limbicsystem (involved in emotions such as rage, fear, motivation sexualbehaviours and feeding) ; medial septum, cingulate cortex, the amygdalaand hippocampal fields (dentate gyrus, CA3, CA2, CA1) and in varioushypothalamic nuclei (often associated with the limbic system). MessengerRNA was also present in regions that have been associated with painperception; the cingulate cortex, insular cortex, and in mid brain andpons structures (e.g. central grey and reticular formation).

EXAMPLE 3 LOCALISATION OF THE θ SUBUNIT IN MONKEY BRAIN BY WESTERN BLOTANALYSIS AND IMMUNOCYTOCHEMISTRY

Antibodies to the human GABA_(A) Theta subunit were generated bysub-cutaneous injection of two New Zealand White rabbits with aglutathione-S-transferase (GST) fusion protein consisting of residues353-595 of the large cytoplasmic loop region of the theta subunit. DNAencoding this region was cloned into the bacterial expression vectorpGEX-2T (Pharmacia), transformed into E. coli DH10B cells (LifeTechnologies), and expression of the fusion protein was carried outusing the Pharmacia protocols. The bacterial cells were incubated on icein STE solution (150 mM NaCl, 10 mM Tris-HCl pH 8, 1 mM EDTA) containing100 μg/ml Lysozyme for 20 min before the addition of N-lauryl sarkosineto 1.5% (w/v). The bacterial slurry was sonicated on ice, and anyinsoluble matter removed by centrifugation. Triton X-100 was added to 3%(v/v) final and the GST-fusion protein purified by glutathione-agaroseaffinity chromatography. Columns were washed extensively with PBS andthe bound protein eluted with 20 mM free glutathione in 150 mM NaCl, 100mM Tris-HCl pH 9, 1 mM EDTA, 1 mM Dithiothreitol. Eluted protein wasconcentrated by precipitation with 5 volumes of cold acetone,resuspended in water, and stored at −70° C. until use.

For western blot analysis tissue samples were removed and dissected outon a glass plate at 4° C. The tissue was homogenised in 50 mM Tris, pH7.5, containing 1 mM PMSF, 1 μM pepstatin A. The homogenate wascentrifuged (2000×g) for 10 minutes and the supernatant was centrifugedat 20,000×g for 45 minutes. The pellet was resuspended in 50 mM Tris andrecentrifuged. The final pellet was resuspended in 50 mM Tris pH 7.4containing protease inhibitors and detergent (Na-deoxycholate:0.25%, 150mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μM pepstatin and leupeptin. Membranepreparations were separated on a 10% Tris tricine polyacrylamide gel andelectrophoretically transferred to nitrocellulose. Nitrocellulose wasblocked with 5% non-fat milk (marvel™)/PBS/Tween (0.5%) for 1 hour atroom temperature. The anti θ subunit antibody was used at aconcentration of 1:500 made up in PBS/Tween/milk at 4° C. overnight,washed and then incubated with anti-rabbit IgG HRP linked (Amersham) at1:1000 in PBS/Tween/milk for one hour at room temperature. The filterswere washed, incubated in ECL (Amersham) for 1 min and opposed to film.A single band of approximately 60-66 kDa was visualised in brainstem andstriatal membranes, close to the predicted molecular weight for the θsubunit of 68-74 kDa.

For localisation of the θ subunit by immunocytochemistry a rhesus monkeywas deeply anesthetised with ketamine and sodium pentobarbitone andtranscardially perfused with saline, followed by 10% formal saline. Thebrain was removed, post fixed for 24 hours, and sliced into coronalblocks, which were then dehydrated through graded alcohols, cleared andembedded in paraffin wax. Coronal sections (8 μm) were cut on a basesledge microtome and mounted on glass microscope slides. Sections weredeparaffinised, rehydrated and rinsed in 0.1M phosphate buffered saline(PBS). In order to enhance the immunoreactivity sections were subjectedto antigen retrieval techniques. Briefly, sections were placed in 0.1Mcitrate buffer pH 6.0 and given two 5 minute bursts at full power in aconventional microwave oven (800 W). Once rinsed in PBS, sections wereincubated in 5% normal goat serum in PBS, for 1 hr to block backgroundstaining. Sections were then incubated overnight at +4° C. in the anti θsubunit rabbit polyclonal antibody (1:1000 diluted in blocking buffer).Immunoreactivity was visualised using the Vector eliltem system (VectorLaboratories, Peterborough, U.K.), followed by development indiaminobenzidine (DAB) (Sigma, U.K.). Sections were counterstained inGill's haematoxylin (Biomen, High Wycombe, U.K.), dehydrated and mountedfor microscopical examination. For comparison, samples of 10% formalinimmersion fixed post mortem human brainstem were processed in anidentical manner. Comparable sections were used to detect θ subunit andtyrosine hydroxylase (Institut Jacques Boy, Reims, France)immunoreactivity by the application of ³⁵S-labeled goat anti rabbitimmunoglobulin 1:100 (Amersham Life Sciences, U.K.) for 1 hr. Slideswere rinsed in distilled water, dehydrated to 95% ethanol, air dried andexposed to Amersham Hyperfilm βmax. Sections used for theimmunofluorescent colocalisation of θ subunit and tyro sine hydroxylasewere pretreated in the same manner, anti θ subunit immunoreactivity wasdetected using firstly a biotinylated anti rabbit; 1:200 (VectorLaboratories) followed by FITC conjugated streptavidin (Sigma, U.K.).The second rabbit polyclonal serum, anti tyrosine hydroxylase, was againvisualised using biotinylated anti rabbit, reacted with Cy3 conjugatedstrepavidin (Sigma, U.K.). Sections were counterstained with Hoescht33258 (0.5 μg/ml). To avoid any crossreactivity of the detectionsystems, sections were placed in boiling distilled water for 5 minutesprior to the application of the second primary antibody and itssubsequent detection. The distribution of the θ subunit immunoreactivityin monkey brain reflected the distribution of the θ mRNA observed by insitu hybridisation studies (Example 2). Labelled neurons were observedof hypothalamic and cortical pyramidal neurones. Significantlabellingwas observed of cells in the brainstem, including thesubstantia nigra pars compacta, ventral and lateral tegmental areas,pigmented neurones of the locus coeruleus and restricted populationwithin the dorsal raphe. Labelling of cell terminals within the caudateputamen was also observed. This distribution was found to closelyresemble the distribution of tyrosine hydroxylase immunoreactivity, amarker of catocholaminergic neurones and their processes, visualised byimmunoautoradiography. θ subunit colocalisation with tyrosinehydroxylase containing neurons was confirmed, using combinationimmunofluorescence. The expression of the θ subunit seen in both thecatocholaminergic neurons of the substantia nigra pars compacta andlocus coeruleus was further substantiated in sections of human postmortem brainstem.

EXAMPLE 4 CONSTRUCTION OF AN LTK⁻CELL LINE EXPRESSING THE THETA RECEPTORSUBUNIT

A chimeric construct of the theta subunit was constructed in themammalian expression vector pcDNA3.1Zeo (Invitrogen) that consisted ofbases −224 to +99 of bovine GABA_(A) α1 gene, a sequence encoding thec-myc epitope tag (residues 410-419 of the human oncogene productc-myc), a cloning site encoding the amino acidsaparagine—serine—glycine, and DNA encoding residues 22-627 of theGABA_(A) θ gene product. This construct was linearised and the DNAtransfected into a clonal population of mouse Ltk⁻ cells that hadpreviously been shown to be stably transfected with the GABA_(A)receptor subunits α₂β₁γ₁ and separately an Ltk⁻ line stably transfectedwith α₂β₃γ₂. The resultant cells were clonally selected with Zeocinselection (100 μg/ml), and screened to verify stable intrgration andexpression of α₂β₁θγ₁ and α₂β₃θγ₂ respectively.

EXAMPLE 5 WHOLE CELL PATCH-CLAMP OF HEK 293 CELLS TRANSIENTLYTRANSFECTED WITH HUMAN GABA-A RECEPTORS

Experiments were performed on HEK 293 cells transiently transfected withhuman cDNA combinations α₂β1 γ1, and α2β1θγ1 (4 μgs of cDNA total percover-slip) using calcium phosphate precipitation (Chen and Okayama,1988) as previously described (Hadingham et al, 1993). Glass cover-slipscontaining the cells in a monolayer culture were transferred to aperspex chamber on the stage of Nikon Diaphot inverted microscope. Cellswere continuously perfused with a solution containing 124 mM NaCl, 2 mMKCl, 2 mM CaCl₂, 1 mM MgCl₂, 1.25 mM KH₂PO₄, 25 mM NaHCO₃, 11 mMD-glucose, at pH 7.2, and observed using phase-contrast optics.Patch-pipettes were pulled with an approximate tip diameter of 2 μm anda resistance of 4MΩ with borosilicate glass and filled with 130 mM CsCl,10 mM HEPES, 10 mM EGTA, 3 mM Mg⁺-ATP, pH adjusted to 7.3 with CsOH.Cells were patch-clamped in whole-cell mode using an Axopatch 200Bpatch-clamp amplifier. Drug solutions were applied by a double-barrelledpipette assembly, controlled by a stepping motor attached to a Priormanipulator, enabling rapid equilibration around the cell. IncreasingGABA concentrations were applied for 2 sec pulses with a 30 sec intervalbetween applications. Non-cumulative concentration-response curves toGABA were constructed. Curves were fitted using a non-linearsquare-fitting program to the equation f(x)=B_(MAX)/[1+(EC₅₀/x)^(n)]where x is the drug concentration, EC₅₀ is the concentration of drugeliciting a half-maximal response and n is the Hill coefficient. EC₅₀'swere analysed by unpaired students t-test.

The GABA EC₅₀ of HEK 293 cells transiently expressing the GABA_(A)receptor subunit combination α₂β₁θγ₁ is significantly lower than that ofHEK 293 cells transiently expressing the GABA_(A) receptor subunitcombination α₂β₁γ₁ (see FIG. 3).

α₂β_(1γ1) α₂β₁θ_(γ1) EC₅₀ 16.7 ± 3.7 nM 62.7 ± 6.7 nM* Slope 1.6 ± 0.21.5 ± 0.1 *p < 0.001

16 1 1884 DNA Homo Sapien 1 atgctgcgag ccgcagtgat cctgctgctc atcaggacctggctcgcgga gggcaactac 60 cccagtccca tcccgaaatt ccacttcgag ttctcctctgctgtgcccga agtcgtcctg 120 aacctcttca actgcaaaaa ttgtgcaaat gaagctgtggttcaaaagat tttggacagg 180 gtgctgtcaa gatacgatgt ccgcctgaga ccgaattttggaggtgcccc tgtgcctgtg 240 agaatatcta tttatgtcac gagcattgaa cagatctcagaaatgaatat ggactacacg 300 atcacgatgt tttttcatca gacttggaaa gattcacgcttagcatacta tgagaccacc 360 ctgaacttga ccctggacta tcggatgcat gagaagttgtgggtccctga ctgctacttt 420 ctgaacagca aggatgcttt cgtgcatgat gtgactgtggagaatcgcgt gtttcagctt 480 cacccagatg gaacggtgcg gtacggcatc cgactcaccactacagcagt ttgttccctg 540 gatctgcata aattccctat ggacaagcag gcctgcaacctggtggtaga gagctatggt 600 tacacggttg aagacatcat attattctgg gatgacaatgggaacgccat ccacatgact 660 gaggagctgc atatccctca gttcactttc ctgggaaggacgattactag caaggaggtg 720 tatttctaca caggttccta catacgcctg atactgaagttccaggttca gagggaagtt 780 aacagctacc ttgtgcaagt ctactggcct actgtcctcaccactattac ctcttggata 840 tcgttttgga tgaactatga ttcctctgca gccagggtgacaattggctt aacttcaatg 900 ctcatcctga ccaccatcga ctcacatctg cgggataagctccccaacat ttcctgtatc 960 aaggccattg atatctatat cctcgtgtgc ttgttctttgtgttcctgtc cttgctggag 1020 tatgtctaca tcaactatct tttctacagt cgaggacctcggcgccagcc taggcgacgc 1080 aggagacccc gaagagtcat tgcccgctac cgctaccagcaagtggtggt aggaaacgtg 1140 caggatggcc tgattaacgt ggaagacgga gtcagctctctccccatcac cccagcgcag 1200 gcccccctgg caagcccgga aagcctcggt tctttgacgtccacctccga gcaggcccag 1260 ctggccacct cggaaagcct cagcccactc acttctctctcaggccaggc ccccctggcc 1320 actggagaaa gcctgagcga tctcccctcc acctcagagcaggcccggca cagctatggt 1380 gttcgcttta atggtttcca ggctgatgac agtattattcctaccgaaat ccgcaaccgt 1440 gtcgaagccc atggccatgg tgttacccat gaccatgaagattccaatga gagcttgagc 1500 tcggatgagc gccatggcca tggccccagt gggaagcccatgcttcacca tggcgagaag 1560 ggtgtgcaag aagcaggctg ggaccttgat gacaacaatgacaagagcga ctgccttgcc 1620 attaaggagc aattcaagtg tgatactaac agtacctggggccttaatga tgatgagctc 1680 gtggcccatg gccaagagaa ggacagtagc tcagagtctgaggatagttg ccccccaagc 1740 cctgggtgct ccttcactga agggttctcc ttcgatctctttaatcctga ctacgtccca 1800 aaggtcgaca agtggtcccg gttcctcttc cctctggcctttgggttgtt caacattgtt 1860 tactgggtat accatatgta ttag 1884 2 627 PRTHomo Sapien 2 Met Leu Arg Ala Ala Val Ile Leu Leu Leu Ile Arg Thr TrpLeu Ala 1 5 10 15 Glu Gly Asn Tyr Pro Ser Pro Ile Pro Lys Phe His PheGlu Phe Ser 20 25 30 Ser Ala Val Pro Glu Val Val Leu Asn Leu Phe Asn CysLys Asn Cys 35 40 45 Ala Asn Glu Ala Val Val Gln Lys Ile Leu Asp Arg ValLeu Ser Arg 50 55 60 Tyr Asp Val Arg Leu Arg Pro Asn Phe Gly Gly Ala ProVal Pro Val 65 70 75 80 Arg Ile Ser Ile Tyr Val Thr Ser Ile Glu Gln IleSer Glu Met Asn 85 90 95 Met Asp Tyr Thr Ile Thr Met Phe Phe His Gln ThrTrp Lys Asp Ser 100 105 110 Arg Leu Ala Tyr Tyr Glu Thr Thr Leu Asn LeuThr Leu Asp Tyr Arg 115 120 125 Met His Glu Lys Leu Trp Val Pro Asp CysTyr Phe Leu Asn Ser Lys 130 135 140 Asp Ala Phe Val His Asp Val Thr ValGlu Asn Arg Val Phe Gln Leu 145 150 155 160 His Pro Asp Gly Thr Val ArgTyr Gly Ile Arg Leu Thr Thr Thr Ala 165 170 175 Val Cys Ser Leu Asp LeuHis Lys Phe Pro Met Asp Lys Gln Ala Cys 180 185 190 Asn Leu Val Val GluSer Tyr Gly Tyr Thr Val Glu Asp Ile Ile Leu 195 200 205 Phe Trp Asp AspAsn Gly Asn Ala Ile His Met Thr Glu Glu Leu His 210 215 220 Ile Pro GlnPhe Thr Phe Leu Gly Arg Thr Ile Thr Ser Lys Glu Val 225 230 235 240 TyrPhe Tyr Thr Gly Ser Tyr Ile Arg Leu Ile Leu Lys Phe Gln Val 245 250 255Gln Arg Glu Val Asn Ser Tyr Leu Val Gln Val Tyr Trp Pro Thr Val 260 265270 Leu Thr Thr Ile Thr Ser Trp Ile Ser Phe Trp Met Asn Tyr Asp Ser 275280 285 Ser Ala Ala Arg Val Thr Ile Gly Leu Thr Ser Met Leu Ile Leu Thr290 295 300 Thr Ile Asp Ser His Leu Arg Asp Lys Leu Pro Asn Ile Ser CysIle 305 310 315 320 Lys Ala Ile Asp Ile Tyr Ile Leu Val Cys Leu Phe PheVal Phe Leu 325 330 335 Ser Leu Leu Glu Tyr Val Tyr Ile Asn Tyr Leu PheTyr Ser Arg Gly 340 345 350 Pro Arg Arg Gln Pro Arg Arg Arg Arg Arg ProArg Arg Val Ile Ala 355 360 365 Arg Tyr Arg Tyr Gln Gln Val Val Val GlyAsn Val Gln Asp Gly Leu 370 375 380 Ile Asn Val Glu Asp Gly Val Ser SerLeu Pro Ile Thr Pro Ala Gln 385 390 395 400 Ala Pro Leu Ala Ser Pro GluSer Leu Gly Ser Leu Thr Ser Thr Ser 405 410 415 Glu Gln Ala Gln Leu AlaThr Ser Glu Ser Leu Ser Pro Leu Thr Ser 420 425 430 Leu Ser Gly Gln AlaPro Leu Ala Thr Gly Glu Ser Leu Ser Asp Leu 435 440 445 Pro Ser Thr SerGlu Gln Ala Arg His Ser Tyr Gly Val Arg Phe Asn 450 455 460 Gly Phe GlnAla Asp Asp Ser Ile Ile Pro Thr Glu Ile Arg Asn Arg 465 470 475 480 ValGlu Ala His Gly His Gly Val Thr His Asp His Glu Asp Ser Asn 485 490 495Glu Ser Leu Ser Ser Asp Glu Arg His Gly His Gly Pro Ser Gly Lys 500 505510 Pro Met Leu His His Gly Glu Lys Gly Val Gln Glu Ala Gly Trp Asp 515520 525 Leu Asp Asp Asn Asn Asp Lys Ser Asp Cys Leu Ala Ile Lys Glu Gln530 535 540 Phe Lys Cys Asp Thr Asn Ser Thr Trp Gly Leu Asn Asp Asp GluLeu 545 550 555 560 Val Ala His Gly Gln Glu Lys Asp Ser Ser Ser Glu SerGlu Asp Ser 565 570 575 Cys Pro Pro Ser Pro Gly Cys Ser Phe Thr Glu GlyPhe Ser Phe Asp 580 585 590 Leu Phe Asn Pro Asp Tyr Val Pro Lys Val AspLys Trp Ser Arg Phe 595 600 605 Leu Phe Pro Leu Ala Phe Gly Leu Phe AsnIle Val Tyr Trp Val Tyr 610 615 620 His Met Tyr 625 3 1884 DNA HomoSapien 3 atgctgcgag ccgcagtgat cctgctgctc atcaggacct ggctcgcggagggcaactac 60 cccagtccca tcccgaaatt ccacttcgag ttctcctctg ctgtgcccgaagtcgtcctg 120 aacctcttca actgcaaaaa ttgtgcaaat gaagctgtgg ttcaaaagattttggacagg 180 gtgctgtcaa gatacgatgt ccgcctgaga ccgaattttg gaggtgcccctgtgcctgtg 240 agaatatcta tttatgtcac gagcattgaa cagatctcag aaatgaatatggactacacg 300 atcacgatgt tttttcatca gacttggaaa gattcacgct tagcatactatgagaccacc 360 ctgaacttga ccctggacta tcggatgcat gagaagttgt gggtccctgactgctacttt 420 ttgaacagca aggatgcttt cgtgcatgat gtgactgtgg agaatcgcgtgtttcagctt 480 cacccagatg gaacggtgcg gtacggcatc cgactcacca ctacagcagcttgttccctg 540 gatctgcata aattccctat ggacaagcag gcctgcaacc tggtggtagagagctatggt 600 tacacggttg aagacatcat attattctgg gatgacaatg ggaacgccatccacatgact 660 gaggagctgc atatccctca gttcactttc ctgggaagga cgattactagcaaggaggtg 720 tatttctaca caggttccta catacgcctg atactgaagt tccaggttcagagggaagtt 780 aacagctacc ttgtgcaagt ctactggcct actgtcctca ccactattacctcttggata 840 tcgttttgga tgaactatga ttcctctgca gccagggtga caattggcttaacttcaatg 900 ctcatcctga ccaccatcga ctcacatctg cgggataagc tccccaacatttcctgtatc 960 aaggccattg atatctatat cctcgtgtgc ttgttctttg tgttcctgtccttgctggag 1020 tatgtctaca tcaactatct tttctacagt cgaggacctc ggcgccagcctaggcgacac 1080 aggagacccc gaagagtcat tgcccgctac cgctaccagc aagtggtggtaggaaacgtg 1140 caggatggcc tgattaacgt ggaagacgga gtcagctctc tccccatcaccccagcgcag 1200 gcccccctgg caagcccgga aagcctcggt tctttgacgt ccacctccgagcaggcccag 1260 ctggccacct cggaaagcct cagcccactc acttctctct caggccaggcccccctggcc 1320 actggagaaa gcctgagcga tctcccctcc acctcagagc aggcccggcacagctatggt 1380 gttcgcttta atggtttcca ggctgatgac agtatttttc ctaccgaaatccgcaaccgt 1440 gtcgaagccc atggccatgg tgttacccat gaccatgaag attccaatgagagcttgagc 1500 tcggatgagc gccatggcca tggccccagt gggaagccca tgcttcaccatggcgagaag 1560 ggtgtgcaag aagcaggctg ggaccttgat gacaacaatg acaagagcgactgccttgcc 1620 attaaggagc aattcaagtg tgatactaac agtacctggg gccttaatgatgatgagctc 1680 atggcccatg gccaagagaa ggacagtagc tcagagtctg aggatagttgccccccaagc 1740 cctgggtgct ccttcactga agggttctcc ttcgatctct ttaatcctgactacgtccca 1800 aaggtcgaca agtggtcccg gttcctcttc cctctggcct ttgggttgttcaacattgtt 1860 tactgggtat accatatgta ttag 1884 4 627 PRT Homo Sapien 4Met Leu Arg Ala Ala Val Ile Leu Leu Leu Ile Arg Thr Trp Leu Ala 1 5 1015 Glu Gly Asn Tyr Pro Ser Pro Ile Pro Lys Phe His Phe Glu Phe Ser 20 2530 Ser Ala Val Pro Glu Val Val Leu Asn Leu Phe Asn Cys Lys Asn Cys 35 4045 Ala Asn Glu Ala Val Val Gln Lys Ile Leu Asp Arg Val Leu Ser Arg 50 5560 Tyr Asp Val Arg Leu Arg Pro Asn Phe Gly Gly Ala Pro Val Pro Val 65 7075 80 Arg Ile Ser Ile Tyr Val Thr Ser Ile Glu Gln Ile Ser Glu Met Asn 8590 95 Met Asp Tyr Thr Ile Thr Met Phe Phe His Gln Thr Trp Lys Asp Ser100 105 110 Arg Leu Ala Tyr Tyr Glu Thr Thr Leu Asn Leu Thr Leu Asp TyrArg 115 120 125 Met His Glu Lys Leu Trp Val Pro Asp Cys Tyr Phe Leu AsnSer Lys 130 135 140 Asp Ala Phe Val His Asp Val Thr Val Glu Asn Arg ValPhe Gln Leu 145 150 155 160 His Pro Asp Gly Thr Val Arg Tyr Gly Ile ArgLeu Thr Thr Thr Ala 165 170 175 Ala Cys Ser Leu Asp Leu His Lys Phe ProMet Asp Lys Gln Ala Cys 180 185 190 Asn Leu Val Val Glu Ser Tyr Gly TyrThr Val Glu Asp Ile Ile Leu 195 200 205 Phe Trp Asp Asp Asn Gly Asn AlaIle His Met Thr Glu Glu Leu His 210 215 220 Ile Pro Gln Phe Thr Phe LeuGly Arg Thr Ile Thr Ser Lys Glu Val 225 230 235 240 Tyr Phe Tyr Thr GlySer Tyr Ile Arg Leu Ile Leu Lys Phe Gln Val 245 250 255 Gln Arg Glu ValAsn Ser Tyr Leu Val Gln Val Tyr Trp Pro Thr Val 260 265 270 Leu Thr ThrIle Thr Ser Trp Ile Ser Phe Trp Met Asn Tyr Asp Ser 275 280 285 Ser AlaAla Arg Val Thr Ile Gly Leu Thr Ser Met Leu Ile Leu Thr 290 295 300 ThrIle Asp Ser His Leu Arg Asp Lys Leu Pro Asn Ile Ser Cys Ile 305 310 315320 Lys Ala Ile Asp Ile Tyr Ile Leu Val Cys Leu Phe Phe Val Phe Leu 325330 335 Ser Leu Leu Glu Tyr Val Tyr Ile Asn Tyr Leu Phe Tyr Ser Arg Gly340 345 350 Pro Arg Arg Gln Pro Arg Arg His Arg Arg Pro Arg Arg Val IleAla 355 360 365 Arg Tyr Arg Tyr Gln Gln Val Val Val Gly Asn Val Gln AspGly Leu 370 375 380 Ile Asn Val Glu Asp Gly Val Ser Ser Leu Pro Ile ThrPro Ala Gln 385 390 395 400 Ala Pro Leu Ala Ser Pro Glu Ser Leu Gly SerLeu Thr Ser Thr Ser 405 410 415 Glu Gln Ala Gln Leu Ala Thr Ser Glu SerLeu Ser Pro Leu Thr Ser 420 425 430 Leu Ser Gly Gln Ala Pro Leu Ala ThrGly Glu Ser Leu Ser Asp Leu 435 440 445 Pro Ser Thr Ser Glu Gln Ala ArgHis Ser Tyr Gly Val Arg Phe Asn 450 455 460 Gly Phe Gln Ala Asp Asp SerIle Phe Pro Thr Glu Ile Arg Asn Arg 465 470 475 480 Val Glu Ala His GlyHis Gly Val Thr His Asp His Glu Asp Ser Asn 485 490 495 Glu Ser Leu SerSer Asp Glu Arg His Gly His Gly Pro Ser Gly Lys 500 505 510 Pro Met LeuHis His Gly Glu Lys Gly Val Gln Glu Ala Gly Trp Asp 515 520 525 Leu AspAsp Asn Asn Asp Lys Ser Asp Cys Leu Ala Ile Lys Glu Gln 530 535 540 PheLys Cys Asp Thr Asn Ser Thr Trp Gly Leu Asn Asp Asp Glu Leu 545 550 555560 Met Ala His Gly Gln Glu Lys Asp Ser Ser Ser Glu Ser Glu Asp Ser 565570 575 Cys Pro Pro Ser Pro Gly Cys Ser Phe Thr Glu Gly Phe Ser Phe Asp580 585 590 Leu Phe Asn Pro Asp Tyr Val Pro Lys Val Asp Lys Trp Ser ArgPhe 595 600 605 Leu Phe Pro Leu Ala Phe Gly Leu Phe Asn Ile Val Tyr TrpVal Tyr 610 615 620 His Met Tyr 625 5 19 DNA Homo Sapien 5 gcaaatgaagctgtggttc 19 6 20 DNA Homo Sapien 6 caatgttgaa caacccaaag 20 7 19 DNAHomo Sapien 7 gcctgagacc gaattttgg 19 8 19 DNA Homo Sapien 8 ggaaccgggaccacttgtc 19 9 18 DNA Homo Sapien 9 tagtccaggg tcaagttc 18 10 20 DNAHomo Sapien 10 tagtatgcta agcgtgaatc 20 11 18 DNA Homo Sapien 11gagtttgagg atagttgc 18 12 18 DNA Homo Sapien 12 tgctccttca ctgaaggg 1813 42 DNA Homo Sapien 13 ccatgactca agcttgccac catgctgcga gccgcagtga tc42 14 27 DNA Homo Sapien 14 tgaaaggagc acagcacagt gctcccg 27 15 45 DNAHomo Sapien 15 ctgcttcttg cacacccttc tcgccatggt gaagcatggg cttcc 45 1645 DNA Homo Sapien 16 tgtcgcctag gctggcgccg aggtcctcga ctgtagaaaa gatag45

What is claimed is:
 1. A stably co-transfected eukaryotic cell linecapable of expressing a human GABA_(A) receptor, which receptorcomprises a θ receptor subunit of SEQ ID NO:4 and at least one receptorsubunit.
 2. A cell line according to claim 1 which is a rodentfibroblast cell line.
 3. A process for the preparation of an eukaryoticcell line capable of expressing a human GABA_(A) receptor, whichcomprises stably co-transfecting a eukaryotic host cell with at leasttwo expression vectors, one such vector comprising the cDNA sequenceencoding the human θ GABA_(A) receptor subunit of SEQ ID NO:4 and onesuch vector comprising the cDNA sequence encoding an α GABA_(A) receptorsubunit.
 4. A process according to claim 3 wherein the cell line is arodent fibroblast cell line.
 5. A recombinant nucleic acid moleculeencoding the θ subunit of the human GABA_(A) receptor comprising asequence selected from the group consisting of the sequence depicted inSEQ ID NO:3; the complement of said sequence, and sequences having atleast 95% homology to SEQ ID NO:3 or its complement, wherein a humanGABA_(A) receptor having the subunit combination α₂β₁θ₁γ₁ has a loweraffinity for GABA as compared to a human GABA_(A) receptor having thesubunit combination α₂β₁θ₁γ₁.
 6. A recombinant expression vectorcomprising a human GABA_(A) receptor θ subunit nucleotide sequenceselected from the group of SEQ ID NO:3, the complement of said sequence,and sequences having 95% homology to SEQ ID NO: 3 or its complement,together with additional sequences capable of directing the synthesis ofsaid human GABA_(A) receptor θ subunit in cultures of transfectedeukaryotic cells, wherein a human GABA_(A) receptor having the subunitcombination α₂β₁θ₁γ₁ has a lower affinity for GABA as compared to ahuman GABA_(A) receptor having the subunit combination α₂β₁θ₁γ₁.
 7. Arecombinantly produced protein preparation comprising a GABA_(A)receptor having subunit combinations comprising a θ receptor subunit ofSEQ ID NO:4 and at least one a receptor subunit.
 8. A recombinantlyproduced membrane preparation comprising a GABA_(A) receptor havingsubunit combinations comprising a θ receptor subunit of SEQ ID NO:4 andat least one α receptor subunit.
 9. A preparation according to claim 7wherein the subunit combination derived is selected from the groupconsisting of the α₁θγ₂, α₂β₁θγ₁ or α₂β₃θγ₂ subunit combination of theGABA_(A) receptor.
 10. A method for determining whether a ligand, notknown to be capable of binding to a human GABA_(A) receptor comprising aθ subunit, can bind to a human GABA_(A) receptor comprising a θ subunit,which comprises (a) contacting a mammalian cell comprising DNA moleculesencoding at least one a receptor subunit and a θ receptor subunit of SEQID NO:4 with the ligand under conditions permitting binding of ligandsknown to bind to the GABA_(A) receptor, (b) detecting the presence orabsence of any of the ligand bound to the GABA_(A) receptor comprising aθ subunit and (c) thereby determining whether the ligand binds to theGABA_(A) receptor comprising a θ subunit.
 11. A method of screeningdrugs to identify drugs which specifically interact with, and bind to, ahuman GABA_(A) receptor comprising a θ subunit on the surface of a cellwhich comprises (a) contacting a mammalian cell comprising a humanGABA_(A) receptor comprising at least one α receptor subunit and a θreceptor subunit of SEQ ID NO:4, on the surface of said cell with aplurality of drugs, (b) determining which of those drugs bind to themammalian cell, and (c) thereby identifying drugs specifically interactwith, and bind to, human GABA_(A) receptors comprising the θ subunit.12. A recombinant GABA_(A) receptor θ subunit polypeptide which has anamino acid sequence depicted in SEQ ID NO:4.
 13. A preparation accordingto claim 8 wherein the subunit combination derived is the α₁θγ₂, α₂β₁θγ₁or α₂β₃θγ₂ subunit combination of the GABA_(A) receptor.
 14. A stablyco-transfected eukaryotic cell line which expresses a human GABA_(A)receptor, which receptor comprises the subunit combination α₂β₁θγ₁,wherein the θ receptor subunit has the sequence of SEQ ID NO:4.
 15. Acell line according to claim 14 which is a rodent fibroblast cell line.16. A process for the preparation of an eukaryotic cell line capable ofexpressing a human GABA_(A) receptor, said receptor comprising thesubunit combination α₂β₁θγ₁, wherein said θ receptor subunit has thesequence depicted in SEQ ID NO:4, said process comprising stablyco-transfecting a eukaryotic host cell with at least two expressionvectors, one such vector comprising the cDNA sequence encoding the humanθ GABA_(A) receptor subunit and one such vector comprising the cDNAsequence encoding an alpha GABA_(A) receptor subunit.
 17. A processaccording to claim 16 wherein the cell line is a rodent fibroblast cellline.
 18. A recombinant nucleic acid molecule encoding the θ subunit ofthe human GABA_(A) receptor, said θ subunit having the sequence depictedin SEQ ID NO:4.
 19. A recombinant expression vector comprising thenucleotide sequence of the human GABA_(A) receptor theta subunitsequence depicted in SEQ ID NO:4, together with additional sequencescapable of directing the synthesis of the said human GABA_(A) receptor θsubunit in cultures of transfected eukaryotic cells.
 20. A recombinantlyproduced protein preparation comprising a GABA_(A) receptor having thesubunit combination α₂β₁θγ₁, wherein said θ receptor subunit has thesequence depicted in SEQ ID NO:4.
 21. A recombinantly produced membranepreparation comprising a GABA_(A) receptor having the subunitcombination α₂β₁θγ₁, wherein said θ receptor subunit has the sequencedepicted in SEQ ID NO:4.
 22. A method for determining whether a ligandnot known to be capable of binding to a human GABA_(A) receptor can bindto said receptor, said receptor having the subunit combination α₂β₁θγ₁₂,wherein said θ receptor subunit has the sequence depicted in SEQ IDNO:4, which method comprises (a) contacting a mammalian cell expressinga human GABA_(A) receptor having the subunit combination α₂β₁θγ₁,wherein said θ receptor subunit has the sequence depicted in SEQ IDNO:4, with the ligand under conditions permitting binding of ligandsknown to bind to the GABA_(A) receptor, (b) detecting the presence orabsence of any of the ligand bound to the GABA_(A) receptor comprisingthe θ subunit and (c) thereby determining whether the ligand binds tothe GABA_(A) receptor.
 23. A method of screening drugs to identify drugswhich specifically interact with, and bind to, a human GABA_(A) receptoron the surface of a cell, the receptor having the subunit combinationα₂β₁θγ₁, wherein said θ receptor subunit has the sequence depicted inSEQ ID NO:4, which method comprises (a) contacting a mammalian cellexpressing a human GABA_(A) receptor having the subunit combinationα₂β₁θγ₁, wherein said θ receptor subunit has the sequence depicted inSEQ ID NO:4, on the surface of said cell with a plurality of drugs, (b)determining which of those drugs bind to the mammalian cell, and (c)thereby identifying drugs specifically interact with, and bind to, thehuman GABA_(A) receptor.
 24. A recombinant GABA_(A) receptor thetasubunit polypeptide which has an amino acid sequence depicted in SEQ IDNO:4.